A cascade signal amplification strategy for the ultrasensitive fluorescence detection of kanamycin base on exonuclease III and mismatched catalytic hairpin assembly.

This study employs a guanine (G)-rich DNA sequence as the recognition element and integrates exonuclease III (Exo III) with a mismatch catalytic hairpin assembly (MCHA)-based cascade isothermal signal amplification strategy to construct a novel fluorescent DNA biosensor for the highly sensitive detection of kanamycin (Kana). In the presence of the target, the recognition element HP1 is unfolded by the target and forms a double-stranded HP1-HP2 structure with HP2. This structure is subsequently cleaved by Exo III, releasing the trigger strand of MCHA. The trigger strand binds to H1, which contains dual fluorescent groups, resulting in the separation of carboxyfluorescein (FAM) and tetramethylrhodamine (TAMRA). This separation attenuates fluorescence resonance energy transfer and restores FAM fluorescence, generating a strong fluorescence signal at 520 nm. The fluorescence sensor demonstrates a linear detection range from 2 to 12 nM, with a detection limit of 0.16 nM. In real milk samples, the spiked recovery rate ranges from 97.4 to 106.3%, with relative standard deviations between 2.2 and 3.8%. The cascade isothermal signal amplification strategy significantly enhances the sensor's sensitivity, while MCHA reduces false positive rates. This aptamer-based sensor exhibits excellent specificity, minimal susceptibility to interference, and suitability for detecting Kana in milk.